Novel low molecular weight cationic lipids for oligonucleotide delivery

ABSTRACT

The instant invention provides for novel catiomc lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver. The present invention employs low molecular weight cationic lipids with one short lipid chain to enhance the efficiency and tolerability of in vivo delivery of siRNA.

BACKGROUND OF THE INVENTION

The present invention relates to novel cationic lipids that can be usedin combination with other lipid components such as cholesterol andPEG-lipids to form lipid nanoparticles with oligonucleotides, tofacilitate the cellular uptake and endosomal escape, and to knockdowntarget mRNA both in vitro and in vivo.

Cationic lipids and the use of cationic lipids in lipid nanoparticlesfor the delivery of oligonucleotides, in particular siRNA and miRNA,have been previously disclosed. Lipid nanoparticles and use of lipidnanoparticles for the delivery of oligonucleotides, in particular siRNAand miRNA, has been previously disclosed. Oligonucleotides (includingsiRNA and miRNA) and the synthesis of oligonucleotides has beenpreviously disclosed. (See US patent applications: US 2006/0083780, US2006/0240554, US 2008/0020058, US 2009/0263407 and US 2009/0285881 andPCT patent applications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 andWO2010/054406). See also Semple S. C. et al., Rational design ofcationic lipids for siRNA delivery, Nature Biotechnology, 2010, 28,172-176.

Other cationic lipids are disclosed in US patent applications: US2009/0263407, US 2009/0285881, US 2010/0055168, US 2010/0055169, US2010/0063135, US 2010/0076055, US 2010/0099738 and US 2010/0104629.

Traditional cationic lipids such as CLinDMA and DLinDMA have beenemployed for siRNA delivery to liver but suffer from non-optimaldelivery efficiency along with liver toxicity at higher doses. It is anobject of the instant invention to provide a cationic lipid scaffoldthat demonstrates enhanced efficacy along with lower liver toxicity as aresult of lower lipid levels in the liver. The present invention employslow molecular weight cationic lipids with one short lipid chain toenhance the efficiency and tolerability of in vivo delivery of siRNA.

SUMMARY OF THE INVENTION

The instant invention provides for novel cationic lipids that can beused in combination with other lipid components such as cholesterol andPEG-lipids to form lipid nanoparticles with oligonucleotides. It is anobject of the instant invention to provide a cationic lipid scaffoldthat demonstrates enhanced efficacy along with lower liver toxicity as aresult of lower lipid levels in the liver. The present invention employslow molecular weight cationic lipids with one short lipid chain toenhance the efficiency and tolerability of in vivo delivery of siRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: LNP (Compound 1) efficacy in mice.

FIG. 2. LNP (Compounds 32 and 33) efficacy in rat (ApoB siRNA).

FIG. 3. Cationic lipid (Compounds 32 and 33) levels in rat liver.

FIG. 4. LNP (Compound 32 and 33, ApoB siRNA) efficacy in NHP.

FIG. 5. LNP (Compound 32 and 33, β-catenin siRNA) efficacy in NHP.

FIG. 6. Peak ALT levels in NHP post LNP dose (Compound 32 and 33).

FIG. 7. Cationic lipid (Compounds 32 and 33) levels in NHP liver.

FIG. 8. Liver β-catenin mRNA KD in TRE-Met mice (Compound 33).

FIG. 9. Tumor β-catenin mRNA KD in TRE-Met mice (Compound 33).

FIG. 10. Tumor growth inhibition (Compound 33) in TRE-met mice.

FIG. 11. LNP (Compounds 32 and 33) efficacy in mice.

FIG. 12. Liver β-catenin mRNA KD in TRE-Met mice (Compound 32).

FIG. 13. Tumor β-catenin mRNA KD in TRE-Met mice (Compound 32).

FIG. 14. Tumor growth inhibition (Compound 32) in TRE-met mice.

DETAILED DESCRIPTION OF THE INVENTION

The various aspects and embodiments of the invention are directed to theutility of novel cationic lipids useful in lipid nanoparticles todeliver oligonucleotides, in particular, siRNA and miRNA, to any targetgene. (See US patent applications: US 2006/0083780, US 2006/0240554, US2008/0020058, US 2009/0263407 and US 2009/0285881 and PCT patentapplications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405 andWO2010/054406). See also Semple S. C. et al., Rational design ofcationic lipids for siRNA delivery, Nature Biotechnology, 2010, 28,172-176.

The cationic lipids of the instant invention are useful components in alipid nanoparticle for the delivery of oligonucleotides, specificallysiRNA and miRNA.

In a first embodiment of this invention, the cationic lipids areillustrated by the Formula A:

wherein:

R¹ and R² are independently selected from H, (C₁-C₆)alkyl, heterocyclyl,and polyamine, wherein said alkyl, heterocyclyl and polyamine areoptionally substituted with one to three substituents selected from R′,or R¹ and R² can be taken together with the nitrogen to which they areattached to form a monocyclic heterocycle with 4-7 members optionallycontaining, in addition to the nitrogen, one or two additionalheteroatoms selected from N, O and S, said monocyclic heterocycle isoptionally substituted with one to three substituents selected from R′;

R³ is independently selected from H and (C₁-C₆)alkyl, said alkyloptionally substituted with one to three substituents selected from R′;

R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ orCON(R″)₂;

R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkylis optionally substituted with halogen and OH;

n is 0, 1, 2, 3, 4 or 5;

L₁ is selected from C₄-C₂₄ alkyl and C₄-C₂₄ alkenyl, said alkyl andalkenyl are optionally substituted with one or more substituentsselected from R′; and

L₂ is selected from C₃-C₉ alkyl and C₃-C₉ alkenyl, said alkyl andalkenyl are optionally substituted with one or more substituentsselected from R′;

or any pharmaceutically acceptable salt or stereoisomer thereof.

In a second embodiment, the invention features a compound having FormulaA, wherein:

R¹ and R² are each methyl;

R³ is H;

n is 0;

L₁ is selected from C₄-C₂₄ alkyl and C₄-C₂₄ alkenyl; and

L₂ is selected from C₃-C₉ alkyl and C₃-C₉ alkenyl;

or any pharmaceutically acceptable salt or stereoisomer thereof.

In a third embodiment, the invention features a compound having FormulaA, wherein:

R¹ and R² are each methyl;

R³ is H;

n is 2;

L₁ is selected from C₄-C₂₄ alkyl and C₄-C₂₄ alkenyl; and

L₂ is selected from C₃-C₉ alkyl and C₃-C₉ alkenyl;

or any pharmaceutically acceptable salt or stereoisomer thereof.

Specific cationic lipids are:

-   (20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine (Compound 1);-   (17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-9-amine (Compound 2);-   (16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-8-amine (Compound 3);-   (13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine (Compound 4);-   (12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine (Compound 5);-   (14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine (Compound 6);-   (15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine (Compound 7);-   (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine (Compound 8);-   (15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine (Compound 9);-   (14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine (Compound 10);-   (19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-9-amine (Compound 11);-   (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine (Compound 12);-   (17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine (Compound 13);-   (16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine (Compound 14);-   (22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine (Compound    15);-   (21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine (Compound 16);-   (18Z)—N,N-dimethylheptacos-18-en-10-amine (Compound 17);-   (17Z)—N,N-dimethylhexacos-17-en-9-amine (Compound 18);-   (19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine (Compound 19); and-   N,N-dimethylheptacosan-10-amine (Compound 20);-   (20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine (Compound    21);-   1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine (Compound 22);-   (20Z)—N,N-dimethylheptacos-20-en-10-amine (Compound 23);-   (15Z)—N,N-dimethylheptacos-15-en-10-amine (Compound 24);-   (14Z)—N,N-dimethylnonacos-14-en-10-amine (Compound 25);-   (17Z)—N,N-dimethylnonacos-17-en-10-amine (Compound 26);-   (24Z)—N,N-dimethyltritriacont-24-en-10-amine (Compound 27);-   (20Z)—N,N-dimethylnonacos-20-en-10-amine (Compound 28);-   (22Z)—N,N-dimethylhentriacont-22-en-10-amine (Compound 29);-   (16Z)—N,N-dimethylpentacos-16-en-8-amine (Compound 30);-   (12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (Compound    31);-   (13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound    32);-   N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine    (Compound 33);-   1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine    (Compound 34);-   N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine    (Compound 35);-   N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine    (Compound 36);-   N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine    (Compound 37);-   N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine    (Compound 38);-   N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine    (Compound 39);-   N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine    (Compound 40)-   1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine    (Compound 41);-   1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine    (Compound 42);-   N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine    (Compound 43); and-   (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,23-trien-10-amine (Compound    44);    or any pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the cationic lipids disclosed are useful in thepreparation of lipid nanoparticles.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of oligonucleotides.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA and miRNA.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA.

The cationic lipids of the present invention may have asymmetriccenters, chiral axes, and chiral planes (as described in: E. L. Elieland S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons,New York, 1994, pages 1119-1190), and occur as racemates, racemicmixtures, and as individual diastereomers, with all possible isomers andmixtures thereof, including optical isomers, being included in thepresent invention. In addition, the cationic lipids disclosed herein mayexist as tautomers and both tautomeric forms are intended to beencompassed by the scope of the invention, even though only onetautomeric structure is depicted.

It is understood that substituents and substitution patterns on thecationic lipids of the instant invention can be selected by one ofordinary skill in the art to provide cationic lipids that are chemicallystable and that can be readily synthesized by techniques known in theart, as well as those methods set forth below, from readily availablestarting materials. If a substituent is itself substituted with morethan one group, it is understood that these multiple groups may be onthe same carbon or on different carbons, so long as a stable structureresults.

It is understood that one or more Si atoms can be incorporated into thecationic lipids of the instant invention by one of ordinary skill in theart to provide cationic lipids that are chemically stable and that canbe readily synthesized by techniques known in the art from readilyavailable starting materials.

In the compounds of Formula A, the atoms may exhibit their naturalisotopic abundances, or one or more of the atoms may be artificiallyenriched in a particular isotope having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberpredominantly found in nature. The present invention is meant to includeall suitable isotopic variations of the compounds of Formula A. Forexample, different isotopic forms of hydrogen (H) include protium (¹H)and deuterium (²H). Protium is the predominant hydrogen isotope found innature. Enriching for deuterium may afford certain therapeuticadvantages, such as increasing in vivo half-life or reducing dosagerequirements, or may provide a compound useful as a standard forcharacterization of biological samples. Isotopically-enriched compoundswithin Formula A can be prepared without undue experimentation byconventional techniques well known to those skilled in the art or byprocesses analogous to those described in the Scheme and Examples hereinusing appropriate isotopically-enriched reagents and/or intermediates.

As used herein, “alkyl” means a straight chain, cyclic or branchedsaturated aliphatic hydrocarbon having the specified number of carbonatoms.

As used herein, “alkenyl” means a straight chain, cyclic or branchedunsaturated aliphatic hydrocarbon having the specified number of carbonatoms including but not limited to diene, triene and tetraeneunsaturated aliphatic hydrocarbons.

Examples of a cyclic “alkyl” or “alkenyl include:

As used herein, “heterocyclyl” or “heterocycle” means a 4- to10-membered aromatic or nonaromatic heterocycle containing from 1 to 4heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocyclyl” therefore includes, thefollowing: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof all of which are optionally substituted with one tothree substituents selected from R″.

As used herein, “polyamine” means compounds having two or more aminogroups. Examples include putrescine, cadaverine, spermidine, andspermine.

As used herein, “halogen” means Br, Cl, F and I.

In an embodiment of Formula A, R¹ and R² are independently selected fromH and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted withone to three substituents selected from R′, or R¹ and R² can be takentogether with the nitrogen to which they are attached to form amonocyclic heterocycle with 4-7 members optionally containing, inaddition to the nitrogen, one or two additional heteroatoms selectedfrom N, O and S, said monocyclic heterocycle is optionally substitutedwith one to three substituents selected from R′.

In an embodiment of Formula A, R¹ and R² are independently selected fromH, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl areoptionally substituted with one to three substituents selected from R′,or R¹ and R² can be taken together with the nitrogen to which they areattached to form a monocyclic heterocycle with 4-7 members optionallycontaining, in addition to the nitrogen, one or two additionalheteroatoms selected from N, O and S, said monocyclic heterocycle isoptionally substituted with one to three substituents selected from R′.

In an embodiment of Formula A, R¹ and R² are independently selected fromH, methyl, ethyl and propyl.

In an embodiment of Formula A, R¹ and R² are each methyl.

In an embodiment of Formula A, R³ is independently selected from: H andmethyl.

In an embodiment of Formula A, R³ is H.

In an embodiment of Formula A, R′ is R″.

In an embodiment of Formula A, R″ is independently selected from H,methyl, ethyl and propyl, wherein said methyl, ethyl and propyl areoptionally substituted with one or more halogen and OH.

In an embodiment of Formula A, R″ is independently selected from H,methyl, ethyl and propyl.

In an embodiment of Formula A, n is 0, 1, 2 or 3.

In an embodiment of Formula A, n is 0, 1 or 2.

In an embodiment of Formula A, n is 0, 1 or 2.

In an embodiment of Formula A, n is 0.

In an embodiment of Formula A, n is 2.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₄ alkyl andC₄-C₂₄ alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₄ alkyl andC₄-C₂₄ alkenyl.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₄ alkenyl.

In an embodiment of Formula A, L₁ is selected from C₁₂-C₂₄ alkenyl.

In an embodiment of Formula A, L₁ is C₁₉ alkenyl.

In an embodiment of Formula A, L₁ is:

In an embodiment of Formula A, L₁ is:

In an embodiment of Formula A, L₂ is selected from C₃-C₉ alkyl and C₃-C₉alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₅-C₉ alkyl and C₅-C₉alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₇-C₉ alkyl and C₇-C₉alkenyl, which are optionally substituted with halogen and OH.

In an embodiment of Formula A, L₂ is selected from C₃-C₉ alkyl and C₃-C₉alkenyl.

In an embodiment of Formula A, L₂ is selected from C₅-C₉ alkyl and C₅-C₉alkenyl.

In an embodiment of Formula A, L₂ is selected from C₇-C₉ alkyl and C₇-C₉alkenyl.

In an embodiment of Formula A, L₂ is C₃-C₉ alkyl.

In an embodiment of Formula A, L₂ is C₅-C₉ alkyl.

In an embodiment of Formula A, L₂ is C₇-C₉ alkyl.

In an embodiment of Formula A, L₂ is C₉ alkyl.

In an embodiment of Formula A, L₁ is selected from C₄-C₂₄ alkyl andC₄-C₂₄ alkenyl, said alkyl and alkenyl are optionally substituted withone or more substituents selected from R′; and L₂ is selected from C₃-C₉alkyl and C₃-C₉ alkenyl, said alkyl and alkenyl are optionallysubstituted with one or more substituents selected from R′.

In an embodiment of Formula A, L₁ is selected from C₁₂-C₂₄ alkenyl, saidalkenyl is optionally substituted with one or more substituents selectedfrom R′; and L₂ is selected from C₅-C₉ alkyl, said alkyl is optionallysubstituted with one or more substituents selected from R′.

In an embodiment of Formula A, L₁ is selected from C₁₋₉ alkenyl, saidalkenyl is optionally substituted with one or more substituents selectedfrom R′; and L₂ is selected from C₇-C₉ alkyl, said alkyl is optionallysubstituted with one or more substituents selected from R′.

In an embodiment of Formula A, L₁ is selected from C₁₋₉ alkenyl, saidalkenyl is optionally substituted with one or more substituents selectedfrom R′; and L₂ is selected from C₉ alkyl, said alkyl is optionallysubstituted with one or more substituents selected from R′.

In an embodiment of Formula A, L₁ is selected from a straight chain C₁₉alkenyl, said alkenyl is optionally substituted with one or moresubstituents selected from R′; and L₂ is selected from a straight chainC₉ alkyl, said alkyl is optionally substituted with one or moresubstituents selected from R′.

In an embodiment of Formula A, “heterocyclyl” is pyrrolidine,piperidine, morpholine, imidazole or piperazine.

In an embodiment of Formula A, “monocyclic heterocyclyl” is pyrrolidine,piperidine, morpholine, imidazole or piperazine.

In an embodiment of Formula A, “polyamine” is putrescine, cadaverine,spermidine or spermine.

In an embodiment, “alkyl” is a straight chain saturated aliphatichydrocarbon having the specified number of carbon atoms.

In an embodiment, “alkenyl” is a straight chain unsaturated aliphatichydrocarbon having the specified number of carbon atoms.

Included in the instant invention is the free form of cationic lipids ofFormula A, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the isolated specific cationic lipidsexemplified herein are the protonated salts of amine cationic lipids.The term “free form” refers to the amine cationic lipids in non-saltform. The encompassed pharmaceutically acceptable salts not only includethe isolated salts exemplified for the specific cationic lipidsdescribed herein, but also all the typical pharmaceutically acceptablesalts of the free form of cationic lipids of Formula A. The free form ofthe specific salt cationic lipids described may be isolated usingtechniques known in the art. For example, the free form may beregenerated by treating the salt with a suitable dilute aqueous basesolution such as dilute aqueous NaOH, potassium carbonate, ammonia andsodium bicarbonate. The free forms may differ from their respective saltforms somewhat in certain physical properties, such as solubility inpolar solvents, but the acid and base salts are otherwisepharmaceutically equivalent to their respective free forms for purposesof the invention.

The pharmaceutically acceptable salts of the instant cationic lipids canbe synthesized from the cationic lipids of this invention which containa basic or acidic moiety by conventional chemical methods. Generally,the salts of the basic cationic lipids are prepared either by ionexchange chromatography or by reacting the free base with stoichiometricamounts or with an excess of the desired salt-forming inorganic ororganic acid in a suitable solvent or various combinations of solvents.Similarly, the salts of the acidic compounds are formed by reactionswith the appropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the cationic lipids of thisinvention include the conventional non-toxic salts of the cationiclipids of this invention as formed by reacting a basic instant cationiclipids with an inorganic or organic acid. For example, conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like, as well as salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,trifluoroacetic (TFA) and the like.

When the cationic lipids of the present invention are acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N¹-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the cationic lipids of the present inventionare potentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof. The reagents utilized in synthesizingthe cationic lipids are either commercially available or are readilyprepared by one of ordinary skill in the art.

Synthesis of the novel cationic lipids is a linear process starting fromlipid acid (I). Coupling to N,O-dimethyl hydroxylamine gives the Weinrebamide II. Grignard addition generates ketone III. Titanium mediatedreductive amination gives final products of type IV.

Synthesis of the single carbon homologated cationic lipids V is a linearprocess starting from lipid ketone (III). Conversion of the ketone tothe nitrile (V) is accomplished via treatment with TOSMIC and potassiumtert-butoxide. Reduction of the nitrile to the primary amine followed byreductive amination provides final cationic lipids VI.

Synthesis of two carbon homologated cationic lipids IX is a linearprocess starting from lipid ketone (III). Conversion of the ketone tothe α,β-unsaturated amide VII is accomplished under Peterson conditions.Conjugate reduction of the α,β-unsaturation is performed usingLS-Selectride to give amide VIII. Reduction of the amide with lithiumaluminum hydride provides final cationic lipids IX.

Cyclopropyl containing lipids are prepared according to General Scheme4. Unsaturated Weinreb amides II are subjected to Simmons-Smithcyclopropanation conditions to give cyclopropyl containing Weinrebamides X. These are carried on to final products as outlined in GeneralSchemes 1-3.

Synthesis of allylic amine cationic lipids XVI is a linear processstarting with aldehyde XI. Addition of t-butyl aceate generatesβ-hydroxy ester XII. Conversion of the hydroxyl functionality to afluoro group followed by acid treatment generates β-fluoro acid XIII.Conversion of the acid to the Weinreb amide followed by Grignardaddition gives the β-fluoro ketone XV. Reductive amination results insimultaneous elimination to generate the desired allylic amine XVI.

20,23-nonacosadien-10-amine, N,N-dimethyl-, (20Z,23Z) (Compound 1)

11,14-Eicosadienoic acid, (11Z,14Z)—(50 g, 162 mmol),N,O-Dimethylhydroxylamine hydrochloride (31.6 g, 324 mmol), HOAt (44.1g, 324 mmol), Et₃N (45.2 mL, 324 mmol), and EDC (62.1 g, 324 mmol) weremixed in DCM (810 mL) and stirred overnight at ambient temperature.Reaction was then washed 5×700 mL water, then washed 1×600 mL 1 M NaOH,dried with sodium sulfate, filtered through celite and evaporated toobtain 53.06 g (93%) 11,14-eicosadienamide, N-methoxy-N-methyl-,(11Z,14Z) as a clear golden oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H),3.68 (s, 3H), 3.18 (s, 3H), 2.77 (m, 2H), 2.41 (t, J=7 Hz, 2H), 2.05 (m,4H), 1.63 (m, 2H), 1.40-1.26 (m, 18H), 0.89 (t, J=7 Hz, 3H).

11,14-eicosadienamide, N-methoxy-N-methyl-, (11Z,14Z)-1 (4 g, 11.38mmol) was dissolved in dry THF (50.0 ml) in a 250 mL flask then 1 Mnonylmagnesium bromide (22.76 ml, 22.76 mmol) was added under nitrogenat ambient temperature. After 10 min, the reaction was slowly quenchedwith excess sat. aq NH₄Cl. The reaction was washed into a separatoryfunnel with hexane and water, shaken, the lower aqueous layer discarded,the upper layer dried with sodium sulfate, filtered, and evaporated togive crude ketone as a golden oil. To the above crude ketone was addeddimethylamine (2 M in THF) (14.22 ml, 28.4 mmol) followed by Ti(O-i-Pr)₄(6.67 ml, 22.76 mmol) and let stir overnight. The next day, added EtOH(50 ml) followed by NaBH₄ (0.646 g, 17.07 mmol). After 5 min ofstirring, directly injected entire reaction onto a 40 g silica columnthat was in line with a 330 g silica column. Eluted 10 min 100% DCM,then 30 min 0-15% MeOH/DCM, collected 20,23-nonacosadien-10-amine,N,N-dimethyl-, (20Z,23Z) (1) (2.45 g, 5.47 mmol, 48.1% yield) as afaintly golden oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H), 2.78 (m,2H), 2.23 (m, 1H), 2.21 (s, 6H), 2.05 (m, 4H), 1.45-1.16 (m, 38H), 0.89(m, 6H). HRMS calcd for C31H61N 448.4877. found 448.4872.

Compounds 2-30 are novel cationic lipids and were prepared according tothe General Scheme 1 above.

Compound Structure HRMS  2

calcd C28H56N 406.4407, found 406.4405.  3

calcd C27H54N 392.4251, found 392.4250.  4

calcd C24H48N 350.3781, found 350.3770.  5

calcd C23H46N 336.3625, found 336.3613.  6

calcd C25H50N 364.3938, found 364.3941.  7

calcd C26H52N 378.4094, found 378.4081.  8

calcd C29H58N 420.4564, found 420.4562.  9

calcd C26H52N 378.4094, found 378.4089. 10

calcd C25H50N 364.3938, found 364.3931. 11

calcd C30H60N 434.4720, found 434.4717. 12

calcd C29H58N 420.4564, found 420.4561. 13

calcd C28H56N 406.4407, found 406.4404. 14

calcd C27H54N 392.4251, found 392.4245. 15

calcd C33H66N 476.5190, found 476.5196. 16

calcd C32H64N 462.5033, found 462.5045. 17

calcd C29H59N 422.4720, found 422.4726. 18

calcd C28H57N 408.4564, found 408.4570. 19

calcd C30H59N 434.4720, found 434.4729. 20

calcd C29H61N 424.4877, found 424.4875. 21

calcd C32H64N 462.5033, found 462.5023. 22

calcd C33H64N 474.5033, found 474.5033. 23

calcd C29H60N 422.4720, found 422.4716. 24

calcd C29H60N 422.4720, found 422.4718. 25

calcd C31H64N 450.5033, found 450.5031. 26

calcd C31H64N 450.5033, found 450.5034. 27

calcd C35H72N 506.5659, found 506.5635. 28

calcd C31H64N 450.5033, found 450.5037. 29

calcd C33H68N 478.5346, found 478.5358. 30

calcd C27H56N 394.4407, found 394.4407.

(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (Compound 31)

A solution of ketone iii (4.0 g, 9.55 mmol), TOSMIC (2.4 g, 12.4 mmol)in dimethoxyethane (45 mL) was cooled to 0° C. and treated withpotassium tert-butoxide (19.1 mmol, 19.1 mL of a 1M solution in tBuOH).After 90 minutes, the reaction was partitioned between hexanes andwater. The organics were washed with water, dried over sodium sulfate,filtered and evaporated in vacuo. This material was purified by flashchromatography (0-5% EtOAc/hexanes) to give desired product (containing˜20% of s.m.). This mixture was carried into next step as is. LC/MS(M+H)=430.6.

Lithium aluminum hydride (23.9 mmol, 23.9 mL of a 1M solution in THF)was added directly to nitrile v (3.42 g, 8 mmol) at ambient temperatureand the reaction was stirred for 20 minutes. The reaction was dilutedwith 100 mL THF, cooled to 0° C. and carefully quenched with sodiumsulfate decahydrate solution. The solids were filtered off and washedwith THF. The filtrate was evaporated in vacuo and carried directly intonext reaction crude. LC/MS (M+H)=434.6.

A solution of primary amine (3.45 g, 6.2 mmol) in dichloroethane (100mL) was treated with formaldehyde (1.6 mL, 21.7 mmol) followed by sodiumtriacetoxyborohydride (6.6 g, 3 mmol). After 5 minutes, the reaction waspartitioned between dichloromethane and 1N NaOH. The organics were driedover sodium sulfate, filtered and evaporated in vacuo. The crude mixturewas purified by reverse phase preparative chromatography (C8 column) toprovide (12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine. HRMScalc'd 462.5033. found 462.5026. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H),2.78 (2H, t, J=5.6 Hz), 2.18 (s, 6H), 2.05 (m, 6H), 1.3 (m, 39H), 0.89(m, 6H).

(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32)

The silyl amide Peterson reagent (3.1 g, 16.7 mmol) was dissolved in THF(35 mL) and cooled to −63° C. To this solution was added nBuLi (16.7mmol, 6.7 mL of a 2.5M solution). The reaction was warmed to ambienttemperature for 30 minutes. The ketone (5.0 g, 11.9 mmol) was dissolvedin THF (25 mL) in a second flask. The ketone solution was transferred tothe Peterson reagent over 30 minutes while maintaining the temperaturebetween −60° C. and −40° C. The reaction was warmed to −40° C. for 1hour, then warmed to 0° C. for 30 minutes. The reaction was quenchedwith sodium bicarbonate, diluted with additional water and partitionedbetween water/hexanes. The organics were washed with brine, dried oversodium sulfate, filtered and evaporated in vacuo. Purification by flashchromatography (0-40% MTBE/hexanes) gave α,β-unsatured amide vii. ¹H NMR(400 MHz, CDCl₃) δ 5.75 (s, 1H), 5.36 (m, 4H), 3.01 (s, 3H), 2.99 (s,3H), 2.78 (t, 2H), 2.28 (t, 2H), 2.05 (m, 6H), 1.35 (m, 34H), 0.89 (m,6H).

α,β-unsatured amide vii (1 g, 2.1 mmol) and LS-Selectride (4.1 mmol, 4.1mL of a 1M solution) were combined in a sealed tube and heated to 60° C.for 24 hours. The reaction was cooled to ambient temperature andpartitioned between ammonium chloride solution and heptane. The organicswere dried over sodium sulfate, filtered and evaporated in vacuo to giveamide viii. This intermediate was carried directly into next reactioncrude.

An alternative conjugate reduction of α,β-unsatured amide vii involvesthe use of a copper hydride reduction:

In a 5 L RB, the Copper catalyst (9.77 g, 17.13 mmol) was dissolved intoluene (1713 ml) under nitrogen. To this was added the PMHS, fromAldrich (304 ml, 1371 mmol) in a single portion. The reaction was agedfor 5 minutes. To the solutions was added the α,β-unsatured amide vii(167.16 g, 343 mmol). To this mixture was then added the t-amyl alcohol(113 ml, 1028 mmol) over 3 h via syringe pump. After addition complete,to the solution was added ˜1700 mL 20% NH4OH to r×n in small portions.Caution: there is vigorous effervescence and foaming in the beginning ofthe quench and it must be closely monitored and the ammonium hydroxideadded slowly in small portions. The reaction was partitioned betweenwater and hexanes. The organics were filtered through celite andevaporated in vacuo. The resulting rubber solid material was pulverizedusing a mechanical stirrer in hexanes to give small particulates whichwere then filtered and washed with hexanes. The organics were thenevaporated in vacuo and purified by flash chromatography (silica, 0-15%ethyl acetate/hexanes) to give desired amide viii. LC/MS (M+H)=490.7.

To a solution of amide viii (2.85 g, 5.8 mmol) was added lithiumaluminum hydride (8.7 mmol, 8.7 mL of a 1M solution). The reaction wasstirred at ambient temperature for 10 minutes then quenched by slowaddition of sodium sulfate decahydrate solution. The solids werefiltered and washed with THF and the filtrate evaporated in vacuo. Thecrude mixture was purified by reverse phase preparative chromatography(C8 column) to provide(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32) asan oil. HRMS (M+H) calc'd 476.5190. found 476.5189. ¹H NMR (400 MHz,CDCl₃) δ 5.37 (m, 4H), 2.78 (t, 2H), 2.42 (m, 8H), 2.05 (q, 4H), 1.28(m, 41H), 0.89 (m, 6H).

N,N-dimethyl-1-(2-octylcyclopropyl)heptadecan-8-amine (Compound 33)

To a solution of oleic acid (1 g, 3.5 mmol) in DCM (500 mL) cooled to 0°C. was added CDI (0.63 g, 3.9 mmol). The reaction was warmed to ambienttemperature for 30 minutes before cooling to 0° C. and treating firstwith triethylamine (0.39 g, 3.9 mmol) and then dimethyl hydroxylaminehydrochloride (0.38 g, 3.9 mmol). After 1 hour the reaction waspartitioned between water and heptane. The organics were dried overmagnesium sulfate, filtered and evaporate in vacuo to give crude Weinrebamide ii which was carried directly into next reaction.

A solution of diethylzinc (70.3 mmol, 70.3 mL of a 1M solution) indichloromethane (130 mL) was cooled to −1° C. and treated dropwise withTFA (8.0 g, 70.3 mmol). After 30 minutes, diiodomethane (18.8 g, 70.3mmol) was added and this was aged for 30 minutes in the ice bath. Tothis solution was added Weinreb amide ii (7.6 g, 23.4 mmol). Thereaction was warmed to ambient temperature and stirred for 1 hour. Thereaction was quenched with ammonium chloride solution (100 mL) andorganic layer partitioned off, washed with 10% sodium thiosulfate, driedover magnesium sulfate, filtered and evaporated in vacuo. Purificationwas flash chromatography (0-30% MTBE/heptane) gave desired product x. ¹HNMR (400 MHz, CDCl₃) δ 3.72 (s, 3H), 3.22 (s, 3H), 2.48 (t, 2H), 1.65(m, 2H), 1.39 (m, 22H), 1.18 (m, 2H), 0.91 (t, 3H), 0.68 (m, 2H), 0.59(m, 1H), −0.32 (m, 1H).

Conversion of Weinreb amide x to Compound 33 was carried out in a manneranalogous to that described for Compound 1 above (nonyl Grignardaddition followed by reductive amination). LC/MS (M+H)=436.6. ¹H NMR(400 MHz, CDCl₃) δ 2.25 (s, 6H), 1.30 (m, 45H), 0.91 (m, 6H), 0.68 (m,2H), 0.59 (m, 1H), −0.31 (m, 1H).

Compounds 34-43 are novel cationic lipids and were prepared according toGeneral Schemes 1-4 above.

Compound Structure HRMS 34

calcd C30H62N 436.4877, found 436.4872. 35

calcd C32H66N 464.5190, found 464.5186. 36

calcd C34H70N 492.5503, found 492.5496. 37

calcd C33H66N 476.5190, found 476.5174. 38

calcd C29H60N 422.4720, found 422.4701. 39

calcd C30H62N 436.4877, found 436.4880. 40

calcd C32H66N 464.5190, found 464.5199. 41

calcd C30H62N 436.4877, found 436.4877. 42

calcd C30H62N 436.4877, found 436.4875. 43

LC/MS (M + H) 408.6.

(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,23-trien-10-amine (Compound 44)

To a solution of LDA (95 mmol, 47.5 mL of a 2M solution) in THF (127 mL)cooled to −78° C. was added t-butyl acetate. The reaction was stirredfor 15 minutes followed by addition of aldehyde xi. The reaction wasimmediately quenched with ammonium chloride solution, warmed to ambienttemperature and partitioned between water/pentane. The organics weredried over sodium sulfate, filtered and evaporated in vacuo. LC/MS(M+H-tBu)=325.4.

Hydroxy ketone xii (7 g, 18.4 mmol) was dissolved in dichloromethane(150 mL) and cooled to 0° C. and treated with deoxofluor (7.3 g, 33.1mmol). The reaction was warmed to ambient temperature with stirring for16 hours followed by quenching with sodium bicarbonate solution. Thereaction was partitioned and the organics dried over sodium sulfate,filtered and evaporate in vacuo. Flash column chromotagraphy (0-5% ethylacetate/hexanes) gave the β-fluoro ester.

Fluoro ester intermediate (6 g, 15.6 mmol) in dichloromethane wastreated with hydrogen chloride (157 mmol, 39.2 mL of a 4M solution indioxane) and the reaction was stirred at ambient temperature for 16hours. The reaction was evaporated in vacuo to give desired β-fluoroacid xiii. LC/MS (M+H)=327.3.

Fluoro carboxylic acid xiii (5.1 g, 15.7 mmol), EDC (6.0 g, 31.4 mmol),N,O-dimethylhydroxylamine hydrochloride (3.1 g, 31.4 mmol),trimethylamine (4.0 g, 39.2 mmol), and HOAt (4.3 g, 31.4 mmol) werecombined in DCM (78 mL) and stirred at ambient temperature for 16 hours.The reaction was partitioned between water/DCM and the organics werewashed with water (3×) and NaOH solution (1×), dried over sodiumsulfate, filtered and evaporated in vacuo. Crude material was purifiedby reverse phase preparative chromatography to give desired Weinrebamide xiv. LC/MS (M+H)=370.4.

A solution of Weinreb amide xiv (4.3 g, 11.7 mmol) in THF (50 mL) wastreated with nonylmagnesium bromide (23.4 mmol, 23.4 mL of a 1Msolution) at ambient temperature. The reaction was quenched withammonium chloride solution after 1 hour and partitioned between waterand pentane. The organics were dried over sodium sulfate, filtered andevaporated in vacuo. This material was carried into next step crude.

Ketone xv (5.1 g, 11.7 mmol) was treated with dimethylamine (29.3 mmol,14.7 mL of a 2M solution in THF) and titanium(IV) isopropoxide (6.7 g,23.5 mmol) and the reaction was stirred at ambient temperature for 16hours. To the reaction mixture was added ethanol (50 mL) followed bysodium borohydride (0.67 g, 17.6 mmol). The reaction was loaded directlyonto a silica column and purified by flash chromatography (0-15%MeOH/DCM). The material required a second purification by preparativereverse phase chromatography to give(11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,23-trien-10-amine. HRMS calc'd446.4720. found 446.4724. ¹H NMR (400 MHz, CDCl₃) δ 5.48 (m, 1H), 5.37(m, 4H), 5.23 (m, 1H), 2.78 (t, 2H), 2.58 (m, 1H), 2.22 (s, 6H), 2.04(m, 6H), 1.56 (m, 1H), 1.30 (m, 31H), 0.89 (m, 6H).

Compound 45 is DLinKC2DMA as described in Nature Biotechnology, 2010,28, 172-176, WO 2010/042877 A1, WO 2010/048536 A2, WO 2010/088537 A2,and WO 2009/127060 A1.

Compound 46 is MC3 as described in WO 2010/054401, and WO 2010/144740A1.

LNP Compositions

The following lipid nanoparticle compositions (LNPs) of the instantinvention are useful for the delivery of oligonucleotides, specificallysiRNA and miRNA:

Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4; CationicLipid/Cholesterol/PEG-DMG 60/38/2; Cationic Lipid/Cholesterol/PEG-DMG67.3/29/3.7; Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7; CationicLipid/Cholesterol/PEG-DMG 50.3/44.3/5.4; CationicLipid/Cholesterol/PEG-C-DMA/DSPC 40/48/2/10; CationicLipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; and CationicLipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10. LNP Process Description:

The Lipid Nano-Particles (LNP) are prepared by an impinging jet process.The particles are formed by mixing lipids dissolved in alcohol withsiRNA dissolved in a citrate buffer. The mixing ratio of lipids to siRNAare targeted at 45-55% lipid and 65-45% siRNA. The lipid solutioncontains a novel cationic lipid of the instant invention, a helper lipid(cholesterol), PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at aconcentration of 5-15 mg/mL with a target of 9-12 mg/mL in an alcohol(for example ethanol). The ratio of the lipids has a mole percent rangeof 25-98 for the cationic lipid with a target of 35-65, the helper lipidhas a mole percent range from 0-75 with a target of 30-50, the PEG lipidhas a mole percent range from 1-15 with a target of 1-6, and the DSPChas a mole percent range of 0-15 with a target of 0-12. The siRNAsolution contains one or more siRNA sequences at a concentration rangefrom 0.3 to 1.0 mg/mL with a target of 0.3-0.9 mg/mL in a sodium citratebuffered salt solution with pH in the range of 3.5-5. The two liquidsare heated to a temperature in the range of 15-40° C., targeting 30-40°C., and then mixed in an impinging jet mixer instantly forming the LNP.The teeID has a range from 0.25 to 1.0 mm and a total flow rate from10-600 mL/min. The combination of flow rate and tubing ID has effect ofcontrolling the particle size of the LNPs between 30 and 200 nm. Thesolution is then mixed with a buffered solution at a higher pH with amixing ratio in the range of 1:1 to 1:3 vol:vol but targeting 1:2vol:vol. This buffered solution is at a temperature in the range of15-40° C., targeting 30-40° C. The mixed LNPs are held from 30 minutesto 2 hrs prior to an anion exchange filtration step. The temperatureduring incubating is in the range of 15-40° C., targeting 30-40° C.After incubating the solution is filtered through a 0.8 um filtercontaining an anion exchange separation step. This process uses tubingIDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000mL/min. The LNPs are concentrated and diafiltered via an ultrafiltrationprocess where the alcohol is removed and the citrate buffer is exchangedfor the final buffer solution such as phosphate buffered saline. Theultrafiltration process uses a tangential flow filtration format (TFF).This process uses a membrane nominal molecular weight cutoff range from30-500 KD. The membrane format can be hollow fiber or flat sheetcassette. The TFF processes with the proper molecular weight cutoffretains the LNP in the retentate and the filtrate or permeate containsthe alcohol; citrate buffer; final buffer wastes. The TFF process is amultiple step process with an initial concentration to a siRNAconcentration of 1-3 mg/mL. Following concentration, the LNPs solutionis diafiltered against the final buffer for 10-20 volumes to remove thealcohol and perform buffer exchange. The material is then concentratedan additional 1-3 fold. The final steps of the LNP process are tosterile filter the concentrated LNP solution and vial the product.

Analytical Procedure:

1) siRNA Concentration

The siRNA duplex concentrations are determined by Strong Anion-ExchangeHigh-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a 2996 PDAdetector. The LNPs, otherwise referred to as RNAi Delivery Vehicles(RDVs), are treated with 0.5% Triton X-100 to free total siRNA andanalyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4×250 mm)column with UV detection at 254 nm. Mobile phase is composed of A: 25 mMNaClO₄, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaClO₄, 10 mM Tris,20% EtOH, pH 7.0 with liner gradient from 0-15 min and flow rate of 1ml/min. The siRNA amount is determined by comparing to the siRNAstandard curve.

2) Encapsulation Rate

Fluorescence reagent SYBR Gold is employed for RNA quantitation tomonitor the encapsulation rate of RDVs. RDVs with or without TritonX-100 are used to determine the free siRNA and total siRNA amount. Theassay is performed using a SpectraMax M5e microplate spectrophotometerfrom Molecular Devices (Sunnyvale, Calif.). Samples are excited at 485nm and fluorescence emission was measured at 530 nm. The siRNA amount isdetermined by comparing to the siRNA standard curve.

Encapsulation rate=(1−free siRNA/total siRNA)×100%

3) Particle Size and Polydispersity

RDVs containing 1 μg siRNA are diluted to a final volume of 3 ml with1×PBS. The particle size and polydispersity of the samples is measuredby a dynamic light scattering method using ZetaPALS instrument(Brookhaven Instruments Corporation, Holtsville, N.Y.). The scatteredintensity is measured with He—Ne laser at 25° C. with a scattering angleof 90°.

4) Zeta Potential Analysis

RDVs containing 1 μg siRNA are diluted to a final volume of 2 ml with 1mM Tris buffer (pH 7.4). Electrophoretic mobility of samples isdetermined using ZetaPALS instrument (Brookhaven InstrumentsCorporation, Holtsville, N.Y.) with electrode and He—Ne laser as a lightsource. The Smoluchowski limit is assumed in the calculation of zetapotentials.

5) Lipid Analysis

Individual lipid concentrations are determined by Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a Corona chargedaerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, Mass.).Individual lipids in RDVs are analyzed using an Agilent Zorbax SB-C18(50×4.6 mm, 1.8 μm particle size) column with CAD at 60° C. The mobilephase is composed of A: 0.1% TFA in H₂O and B: 0.1% TFA in IPA. Thegradient changes from 60% mobile phase A and 40% mobile phase B fromtime 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40%mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobilephase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phaseB from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60%mobile phase A and 40% mobile phase B at 20.00 min with flow rate of 1ml/min. The individual lipid concentration is determined by comparing tothe standard curve with all the lipid components in the RDVs with aquadratic curve fit. The molar percentage of each lipid is calculatedbased on its molecular weight.

General LNP Process Description for Compound 32 Formulations:

The lipid nanoparticles were prepared by an impinging jet process. Theparticles were formed by mixing lipids dissolved in alcohol with siRNAdissolved in a citrate buffer. The lipid solution contained a cationiclipid, a helper lipid (cholesterol), PEG (e.g. PEG-C-DMA, PEG-DMG)lipid, and DSPC at a concentration of 5-15 mg/mL with a target of 9-12mg/mL in an alcohol (for example ethanol). The ratio of the lipids had amole percent range of 25-98 for the cationic lipid with a target of35-65, the helper lipid had a mole percent range from 0-75 with a targetof 30-50, the PEG lipid has a mole percent range from 1-15 with a targetof 1-6, and the DSPC had a mole percent range of 0-15 with a target of0-12. The siRNA solution contained one or more siRNA sequences at aconcentration range from 0.3 to 0.6 mg/mL with a target of 0.3-0.9 mg/mLin a sodium citrate buffered salt solution with pH in the range of3.5-5. The two solutions were heated to a temperature in the range of15-40° C., targeting 30-40° C., and then mixed in an impinging jet mixerinstantly forming the LNP. The teeID had a range from 0.25 to 1.0 mm anda total flow rate from 10-600 mL/minute. The combination of flow rateand tubing ID had the effect of controlling the particle size of theLNPs between 30 and 200 nm. The LNP suspension was then mixed with abuffered solution at a higher pH with a mixing ratio in the range of 1:1to 1:3 vol:vol, but targeting 1:2 vol:vol. This buffered solution was ata temperature in the range of 15-40° C., targeting 30-40° C. This LNPsuspension was further mixed with a buffered solution at a higher pH andwith a mixing ratio in the range of 1:1 to 1:3 vol:vol, but targeting1:2 vol:vol. The buffered solution was at a temperature in the range of15-40° C., targeting 30-40° C. The mixed LNPs were held from 30 minutesto 2 hrs prior to an anion exchange filtration step. The temperatureduring incubating was in the range of 15-40° C., targeting 30-40° C.After incubating, the LNP suspension was filtered through a 0.8 umfilter containing an anion exchange separation step. This process usedtubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to2000 mL/minute The LNPs were concentrated and diafiltered via anultrafiltration process where the alcohol was removed and the citratebuffer was exchanged for the final buffer solution such as phosphatebuffered saline. The ultrafiltration process used a tangential flowfiltration format (TFF). This process used a membrane nominal molecularweight cutoff range from 30-500 KD. The membrane format was hollow fiberor flat sheet cassette. The TFF processes with the proper molecularweight cutoff retained the LNP in the retentate and the filtrate orpermeate contained the alcohol; citrate buffer; and final buffer wastes.The TFF process is a multiple step process with an initial concentrationto a siRNA concentration of 1-3 mg/mL. Following concentration, the LNPsuspension was diafiltered against the final buffer for 10-20 volumes toremove the alcohol and perform buffer exchange. The material was thenconcentrated an additional 1-3 fold. The final steps of the LNP processwere to sterile filter the concentrated LNP solution and vial theproduct.

Analytical Procedure:

siRNA Concentration

The siRNA duplex concentrations were determined by Strong Anion-ExchangeHigh-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a 2996 PDAdetector. The LNPs, otherwise referred to as RNAi Delivery Vehicles(RDVs), were treated with 0.5% Triton X-100 to free total siRNA andanalyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4×250 mm)column with UV detection at 254 nm. Mobile phase was composed of A: 25mM NaClO₄, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaClO₄, 10 mMTris, 20% EtOH, pH 7.0 with a liner gradient from 0-15 min and a flowrate of 1 ml/minute. The siRNA amount was determined by comparing to thesiRNA standard curve.

Encapsulation Rate

Fluorescence reagent SYBR Gold was employed for RNA quantitation tomonitor the encapsulation rate of RDVs. RDVs with or without TritonX-100 were used to determine the free siRNA and total siRNA amount. Theassay is performed using a SpectraMax M5e microplate spectrophotometerfrom Molecular Devices (Sunnyvale, Calif.). Samples were excited at 485nm and fluorescence emission was measured at 530 nm. The siRNA amount isdetermined by comparing to an siNA standard curve.

Encapsulation rate=(1−free siNA/total siNA)×100%

Particle Size and Polydispersity

RDVs containing 1 μg siRNA were diluted to a final volume of 3 ml with1×PBS. The particle size and polydispersity of the samples was measuredby a dynamic light scattering method using ZetaPALS instrument(Brookhaven Instruments Corporation, Holtsville, N.Y.). The scatteredintensity was measured with He—Ne laser at 25° C. with a scatteringangle of 90°.

Zeta Potential Analysis

RDVs containing 1 μg siRNA were diluted to a final volume of 2 ml with 1mM Tris buffer (pH 7.4). Electrophoretic mobility of samples wasdetermined using ZetaPALS instrument (Brookhaven InstrumentsCorporation, Holtsville, N.Y.) with electrode and He—Ne laser as a lightsource. The Smoluchowski limit was assumed in the calculation of zetapotentials.

Lipid Analysis

Individual lipid concentrations were determined by Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a Corona chargedaerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, Mass.).Individual lipids in RDVs were analyzed using an Agilent Zorbax SB-C18(50×4.6 mm, 1.8 μm particle size) column with CAD at 60° C. The mobilephase was composed of A: 0.1% TFA in H₂O and B: 0.1% TFA in IPA. Thegradient changed from 60% mobile phase A and 40% mobile phase B fromtime 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40%mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobilephase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phaseB from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60%mobile phase A and 40% mobile phase B at 20.00 min with a flow rate of 1ml/minute. The individual lipid concentration was determined bycomparing to the standard curve with all the lipid components in theRDVs with a quadratic curve fit. The molar percentage of each lipid wascalculated based on its molecular weight.

General LNP Preparation for Various Formulations in Table 1.

siRNA nanoparticle suspensions in Table 1 are prepared by dissolvingsiRNAs and/or carrier molecules in 20 mM sodium citrate buffer (pH 5.0)at a concentration of about 0.40 mg/mL. Lipid solutions are prepared bydissolving a mixture of cationic lipid (e.g., 32, see structure in Table2), DSPC, Cholesterol, and PEG-DMG (ratios shown in Table 1) in absoluteethanol at a concentration of about 8 mg/mL. The nitrogen to phosphateratio is approximate to 6:1.

Nearly equal volumes of siRNA/carrier and lipid solutions are deliveredwith two FPLC pumps at the same flow rates to a mixing T connector. Aback pressure valve is used to adjust to the desired particle size. Theresulting milky mixture is collected in a sterile glass bottle. Thismixture is then diluted with an equal volume of citrate buffer, followedby equal volume of PBS (pH 7.4), and filtered through an ion-exchangemembrane to remove any free siRNA/carrier in the mixture. Ultrafiltration against PBS (7.4)) is employed to remove ethanol and toexchange buffer. The final LNP is obtained by concentrating to a desiredvolume and sterile filtered through a 0.2 μm filter. The obtained LNPsare characterized in term of particle size, Zeta potential, alcoholcontent, total lipid content, nucleic acid encapsulated, and totalnucleic acid concentration.

LNP Manufacture Process

In a non-limiting example, LNP is prepared in bulk as follows. Theprocess consists of (1) preparing a lipid solution; (2) preparing ansiRNA/carrier solution; (3) mixing/particle formation; (4) incubation;(5) dilution; (6) ultrafiltration and concentration.

Preparation of Lipid Solution

2 L glass reagent bottles and measuring cylinders are depyrogenated. Thelipids are warmed to room temperature. Into the glass reagent bottle istransferred 8.0 g of Compound 32 with a pipette and 1.2 g of DSPC, 3.5 gof Cholesterol, 0.9 g of PEG-DMG are added. To the mixture is added 1 Lof ethanol. The reagent bottle is placed in heated water bath, at atemperature not exceeding 50° C. The lipid suspension is stirred with astir bar. A thermocouple probe is put into the suspension through oneneck of the round bottom flask with a sealed adapter. The suspension isheated at 30-40° C. until it became clear. The solution is allowed tocool to room temperature.

Preparation of siRNA/Carrier Solution

Into a sterile container, such as the Corning storage bottle, is weighed0.4 g times the water correction factor (approximately 1.2) of siRNA-1powder. The siRNA is transferred to a depyrogenated 2 L glass reagentbottle. The weighing container is rinsed 3× with citrate buffer (20 mM,pH 5.0) and the rinses are placed into the 2 L glass bottle, QS withcitrate buffer to 1 L. The concentration of the siRNA solution isdetermined with a UV spectrometer using the following procedure. 20 μLis removed from the solution, diluted 50 times to 1000 μL, and the UVreading recorded at A260 nm after blanking with citrate buffer. This isrepeated. If the readings for the two samples are consistent, an averageis taken and the concentration is calculated based on the extinctioncoefficients of the siRNAs. If the final concentration is out of therange of 0.40±0.01 mg/mL, the concentration is adjusted by adding moresiRNA/carrier powder, or adding more citrate buffer. This process isrepeated for the second siRNA, if applicable.

Alternatively, if the siRNA/carrier solution comprised a single siRNAduplex and/or carrier instead of a cocktail of two or more siRNAduplexes and/or carriers, then the siRNA/carrier is dissolved in 20 mMcitrate buffer (pH 5.0) to give a final concentration of 0.4 mg/mL.

The lipid and ethanol solutions are then sterile filtered through a PallAcropak 0.8/0.2 μm sterile filter PN 12203 into a depyrogenated glassvessel using a Master Flex Peristaltic Pump Model 7520-40 to provide asterile starting material for the encapsulation process. The filtrationprocess is run at an 80 mL scale with a membrane area of 20 cm². Theflow rate is 280 mL/minute. This process is scaleable by increasing thetubing diameter and the filtration area.

Particle Formation—Mixing Step

Using a two-barrel syringe driven pump (Harvard 33 Twin Syringe), thesterile lipid/ethanol solution and the sterile siRNA/carrier orsiRNA/carrier cocktail/citrate buffer (20 mM citrate buffer, pH 5.0)solutions are mixed in a 0.5 mm ID T-mixer (Mixing Stage I) at equal, ornearly equal, flow rates. The resulting outlet LNP suspension contained40-50 vol % ethanol. When a 45 vol % ethanol outlet suspension isdesired, the sterile lipid/ethanol and the sterile siRNA/carrier orsiRNA/carrier cocktail/citrate buffer solutions are mixed at flow ratesof 54 mL/min and 66 mL/min, respectively, such that the total flow rateof the mixing outlet is 120 mL/min.

Dilution

The outlet stream of Mixing Stage I is fed directly into a 4 mm IDT-mixer (Mixing Stage II), where it is diluted with a buffered solutionat higher pH (20 mM sodium citrate, 300 mM sodium chloride, pH 6.0) at aratio of 1:1 vol:vol %. This buffered solution is at a temperature inthe range of 30-40° C., and is delivered to the 4 mm T-mixer via aperistaltic pump (Cole Parmer MasterFlex L/S 600 RPM) at a flow rate of120 mL/min.

The outlet stream of Mixing Stage II is fed directly into a 6 mm IDT-mixer (Mixing Stage III), where it is diluted with a buffered solutionat higher pH (PBS, pH 7.4) at a ratio of 1:1 vol:vol %. This bufferedsolution is at a temperature in the range of 15-25° C., and is deliveredto the 6 mm T-mixer via peristaltic pump (Cole Parmer MasterFlex L/S 600RPM) at a flow rate of 240 mL/min.

Incubation and Free siRNA Removal

The outlet stream of Mixing Stage III is held after mixing for 30 minuteincubation. The incubation is conducted at temperature of 35-40° C. andthe in-process suspension was protected from light. Followingincubation, free (un-encapsulated) siRNA is removed via anion exchangewith Mustang Q chromatography filters (capsules). Prior to use, thechromatography filters are pre-treated sequentially with flushes of 1NNaOH, 1M NaCl, and a final solution of 12.5 vol % ethanol in PBS. The pHof the final flush is checked to ensure pH<8. The incubated LNP streamis then filtered via Mustang Q filters via peristaltic pump (Cole ParmerMasterFlex L/S 600 RPM) at flow rate of approximately 100 mL/min. Thefiltered stream is received into a sterile glass container forultrafiltration and concentration as follows.

Ultrafiltration, Concentration and Sterile Filtration

The ultrafiltration process is a timed process and the flow rates mustbe monitored carefully. This is a two step process; the first is aconcentration step taking the diluted material and concentratingapproximately 8-fold, to a concentration of approximately 0.3-0.6 mg/mLsiRNA.

In the first step, a ring-stand with an ultrafiltration membrane 100 kDaPES (Spectrum Labs) installed is attached to a peristaltic pump(Spectrum KrosFloII System). 9.2 L of sterile distilled water is addedto the reservoir; 3 L is drained to waste and the remainder is drainedthrough permeate to waste. 5.3 L of 0.25 N sodium hydroxide is added tothe reservoir with 1.5 L drained to waste and 3.1 L drained throughpermeate to waste. The remaining sodium hydroxide is held in the systemfor sanitization (at least 10 minutes), and then the pump is drained.9.2 L of 70 (v/v) % isopropyl alcohol is added to the reservoir with 1.5L drained to waste and the remainder drained through permeate to waste.6 L of conditioning buffer (12.5% ethanol in phosphate buffered saline)is added with 1.5 L drained to waste and the remainder drained thoughthe permeate until the waste is of neutral pH (7-8). A membrane fluxvalue is recorded, and the pump is then drained.

The diluted LNP solution is placed into the reservoir to the 1.1 L mark.The pump is turned on at 2.3 L/min. After 5 minutes of recirculation,the permeate pump is turned on at 62.5 mL/min and the liquid level isconstant at approximately 950 mL in the reservoir. The diluted LNPsolution is concentrated from 9.8 L to 1.1 L in 140 minutes, and thepump is paused when all the diluted LNP solution has been transferred tothe reservoir.

The second step is a diafiltration step exchanging the ethanol/aqueousbuffer to phosphate buffered saline. During this step, approximately10-20 diafiltration volumes of phosphate buffered saline are used.Following diafiltration, a second concentration is undertaken toconcentrate the LNP suspension 3-fold to approximately 1-1.5 mg/mLsiRNA. The concentrated suspension is collected into sterile, plasticPETG bottles. The final suspension is then filtered sequentially viaPall 0.45 um PES and Pall 0.2 um PES filters for terminal sterilizationprior to vial filling.

In an embodiment, an LNP composition of the instant invention comprises,a cationic lipid of Formula A, cholesterol, DSPC and PEG-DMG.

In another embodiment, an LNP composition of the instant inventionfurther comprises a cryoprotectant.

In another embodiment, the cryoprotectant is Sucrose, Trehalose,Raffinose, Stachyose, Verbascose, Mannitol, Glucose, Lactose, Maltose,Maltotriose-heptaose, Dextran, Hydroxyethyl Starch, Insulin, Sorbitol,Glycerol, Arginine, Histidine, Lysine, Proline, Dimethylsulfoxide or anycombination thereof.

In another embodiment, the cryoprotectant is Sucrose.

In another embodiment, the cryoprotectant is Trehalose.

In another embodiment, the cryoprotectant is a combination of Sucroseand Trehalose.

In another embodiment, the LNP composition comprises, the cationic lipid(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32),cholesterol, DSPC and PEG-DMG.

The obtained LNPs are characterized in terms of particle size, Zetapotential, alcohol content, total lipid content, nucleic acidencapsulated, and total nucleic acid concentration.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein, as presently representative ofpreferred embodiments, are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

TABLE 1 Composition of Select Lipid Nanoparticle Formulations LNPIdentifier Lipid Components and Molar Ratios siRNA N/P 32 CholesterolDSPC PEG- SEQ ID 5/6 6 (58%) (30%) (10%) DMG (2%) 32 Cholesterol DSPCPEG- SEQ ID 7/8 6 (58%) (30%) (10%) DMG (2%) 32 Cholesterol DSPC PEG-SEQ ID 9/10 6 (58%) (30%) (10%) DMG (2%)

TABLE 2 Chemical Structures of Lipids in Formulations of Table 1. LipidChemical Structure 32

Cholesterol

DSPC

PEG-DMG

Utilizing the above described LNP process, specific LNPs with thefollowing ratios were identified:

Nominal Composition: Cationic Lipid/Cholesterol/PEG-DMG 60/38/2 CationicLipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10

Luc siRNA

(SEQ. ID. NO.: 1) 5′-iB-A U AAGG CU A U GAAGAGA U ATT-iB 3′(SEQ. ID. NO.: 2) 3′-UUUAUUCCGAUACUUCUC UAU-5′ AUGC—RiboseiB—Inverted deoxy abasic UC—2′ Fluoro AGT—2′ Deoxy AGU—2′ OCH₃

Nominal Composition Cationic Lipid/Cholesterol/PEG-DMG 60/38/2 CationicLipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10 CationicLipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10

ApoB siRNA

(SEQ ID NO.: 3) 5′-iB-CUUUAACAAUUCCUGAAAUTsT-iB-3′ (SEQ ID NO.: 4)3′-UsUGAAAUUGUUAAGGACUsUsUsA-5′ AUGC—Ribose iB—Inverted deoxy abasicUC—2′ Fluoro AGT—2′ Deoxy AGU—2′ OCH₃ UsA—phophorothioate linkageBeta-Catenin siRNA

(SEQ ID NO.: 5) 5′-iB-CUGUUGGAUUGAUUCGAAAUsU-iB-3′ (SEQ ID NO.: 6)3′-UsUG ACA A CCUAA C UAAGCUUU-5′ AUGC—Ribose iB—Inverted deoxy abasicUC—2′ Fluoro AGT—2′ Deoxy AGU—2′ OCH₃ UsA—phophorothioate linkage(SEQ ID NO.: 7) 5′-iB-A C GACUA GUUC AGU U G CUUUsU-iB-3′(SEQ ID NO.: 8) 3′-UsUUGCUGAUCAAGUC A ACGAA-5′ AUGC—RiboseiB—Inverted deoxy abasic UC—2′ Fluoro AGT—2′ Deoxy AGU—2′ OCH₃UsA—phophorothioate linkage (SEQ ID NO.: 9) 5′-iB-A C GACUA GUUC AGU U GCUUUU-iB-3′ (SEQ ID NO.: 10) 3′-UUUGCUGAUCAAGUC A ACGAA-5′ AUGC—RiboseiB—Inverted deoxy abasic UC—2′ Fluoro AGT—2′ Deoxy AGU—2′ OCH₃UsA—phophorothioate linkage

Oligonucleotide synthesis is well known in the art. (See US patentapplications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US2009/0263407 and US 2009/0285881 and PCT patent applications: WO2009/086558, WO2009/127060, WO2009/132131, WO2010/042877, WO2010/054384,WO2010/054401, WO2010/054405 and WO2010/054406). The siRNAs disclosedand utilized in the Examples were synthesized via standard solid phaseprocedures.

Example 1 Mouse in Vivo Evaluation of Efficacy

LNPs utilizing Compounds 1-44, in the nominal compositions describedimmediately above, were evaluated for in vivo efficacy. The siRNAtargets the mRNA transcript for the firefly (Photinus pyralis)luciferase gene (Accession # M15077). The primary sequence and chemicalmodification pattern of the luciferase siRNA is displayed above. The invivo luciferase model employs a transgenic mouse in which the fireflyluciferase coding sequence is present in all cells.ROSA26-LoxP-Stop-LoxP-Luc (LSL-Luc) transgenic mice licensed from theDana Farber Cancer Institute are induced to express the Luciferase geneby first removing the LSL sequence with a recombinant Ad-Cre virus(Vector Biolabs). Due to the organo-tropic nature of the virus,expression is limited to the liver when delivered via tail veininjection. Luciferase expression levels in liver are quantitated bymeasuring light output, using an IVIS imager (Xenogen) followingadministration of the luciferin substrate (Caliper Life Sciences).Pre-dose luminescence levels are measured prior to administration of theRDVs. Luciferin in PBS (15 mg/mL) is intraperitoneally (IP) injected ina volume of 150 μL. After a four minute incubation period mice areanesthetized with isoflurane and placed in the IVIS imager. The RDVs(containing siRNA) in PBS vehicle were tail vein injected in a volume of0.2 mL. Final dose levels ranged from 0.1 to 0.5 mg/kg siRNA. PBSvehicle alone was dosed as a control. Mice were imaged 48 hours postdose using the method described above. Changes in luciferin light outputdirectly correlate with luciferase mRNA levels and represent an indirectmeasure of luciferase siRNA activity. In vivo efficacy results areexpressed as % inhibition of luminescence relative to pre-doseluminescence levels. Systemic administration of the luciferase siRNARDVs decreased luciferase expression in a dose dependant manner. Greaterefficacy was observed in mice dosed with Compound 1 containing RDVs thanwith the RDV containing the octyl-CLinDMA (OCD) cationic lipid (FIG. 1).OCD is known and described in WO2010/021865. Similar efficacy wasobserved in mice dosed with Compound 32 and 33 containing RDVs relativeto the RDV containing the MC3 (Compound 46) cationic lipid (FIG. 11).

Example 2 In Vitro ApoE Binding Assay

LNPs are incubated at 37° C. in 90% rhesus serum at a final LNPconcentration of 4 ug/mL. Incubation is for 20 minutes with orbitalrotation. After incubation, the samples are diluted 1:20 in PBS and 100uL of each diluted sample is aliquoted to wells of an anti-PEG antibodycoated 96-well plate (Life Diagnostics Cat. No. P-0001PL. Afterincubation at room temperature for 1 hour, the plate is washed 5× with300 uL PBS. After washing, 50 uL of 0.2% Triton X-100 is added to eachwell and the plate incubated at 37° C. for 10 minutes, followed byshaking on a plate shaker for 1 minute at 750 rpm. Samples are frozenprior to performing the ApoE ELISA and stem loop PCR analysis ofsamples.

An ApoE ELISA assay is performed to quantitate ApoE bound to the LNPsafter incubation in rhesus serum. Anti-ApoE antibody (Milipore, Cat No.AB947) is diluted 1:1000 in PBS and 100 uL of diluted antibody is addedto each well of a polystyrene high binding plate. The plate withantibody is incubated overnight at 4° C., after which the plate iswashed 2× with 200 uL of PBS. Next, 200 uL of buffer containing 1% BSAand 0.05% Tween-20 in PBS (Incubation Buffer) is added to each wellfollowed by incubation at room temperature for 1 hour. Plates are washed5× with PBS containing 0.05% Tween-20. Frozen Triton lysis test samplesare thawed and diluted 1:6 with incubation buffer and 100 uL of testsample is aliquoted to wells of the ApoE antibody plate. Incubation isfor 1 hour at room temperature followed by a 5× wash with PBS containing0.05% Tween-20. After washing, 100 uL of biotinylated anti-ApoE antibody(Mabtech, Cat. ANo. E887-biotin), diluted 1:500 in incubation buffer, isadded to each well and incubated for 1 hour at room temperature,followed by a 5× wash with 0.05% Tween-20 in PBS. 100 uL per well, ofStreptavidin-HPR (Thermo, Cat. No. TS-125-HR), is then added andincubated for 1 hour at room temperature. After washing 5× with 0.05%Tween-20 in PBS, 100 uL of TMB Substrate (Thermo, Cat. No. 34028) isadded to each well, followed by incubation at room temperature for 20minutes in the dark. The colorimetric reaction is stopped with 100 uL ofTMB Stop Solution (KPL, Cat. No. 50-85-04) and absorbance at 450 nm isdetermined. An ApoE standard curve is prepared by diluting rhesusRecombinant ApoE in incubation buffer with 0.03% Triton X-100 withconcentrations ranging from 100 ng/mL to 0.78 ng/mL. ApoE standards areevaluated in the ELISA in parallel to the test samples. A rhesus serumonly (no LNP) control is utilized to obtain a background subtraction fornon-LNP dependent ApoE signal in the ELISA.

Stem Loop RT-PCR Protocol

To normalize to the ApoE bound to the amount of LNP bound to theanti-PEG antibody plate, the amount of siRNA retained in the anti-PEGantibody well is quantitated by stem-loop PCR and related to the numberof siRNAs encapsulated per LNP, to give an approximate measure of totalLNP particles bound per well.

Preparation of the Spiked Standard Curve Samples:

The standard curve is prepared using the molecular weight of the siRNA(13693 g/mol for ApoB 17063) to calculate the copy number. The highstandard should contain 10¹¹ copies per 3 μl. A 10-fold serial dilutionis performed across a row of an assay plate until the lowest standardcontains 10² copies per 3 μl. Dilute 0.2% Triton X-100 1:80 in water andpipette 20 uL of the diluted Triton X-100 into 10 wells of a 96 wellplate. 30 uL of the serial diluted standard curve and mix are added toeach well of the plate. 10 uL of the spiked standard curve is used inthe reverse transcription reaction.

Stem-Loop RT-PCR—Test Samples and Standard Curve:

Triton lysates from the PEG antibody plate capture are diluted 1 to 2000in nuclease free water. 10 uL of ‘RT-Primer Mix’ (Applied Biosystem'sTaqMan MicroRNA Reverse Transcription Kit Cat. No. 4366596) is added toeach well of a 96-well Micro-Amp QPCR plate (ABI Cat# N801-0560).

Final RT Primer Mix Components uL/rxn conc. ApoB RT-primer (10 uM) 0.6200 nM 10x buffer 2 Water 7.4 ApoB RT primer sequence: 5′GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTTTAACA3′ (SEQ. ID. NO.:11)

10 uL of each test sample (diluted 1 to 2000) or spiked standard curve(above) are aliquoted into the 96-well plate. The plate is covered witha mat (ABI Cat. No. N801-0550), to minimize evaporation. The plate isbriefly centrifuged at 800 rpm for 1 minute. Next, the plate is run on athermocycler using the following cycling parameters:

Cycling: 94° C. 10 minutes  75° C. 2 minutes 60° C. 3 minutes 50° C. 3minutes 40° C. 3 minutes 30° C. 3 minutes  4° C. hold

Next, 10 uL of ‘RT Mix’ is added to each well (Applied Biosystem'sTaqMan MicroRNA Reverse Transcription Kit Cat. No. 4366596)

RT Mix Components uL/rxn 100 mM dNTP 0.3 10x RT buffer 1 Rnase Inhibitor0.38 Multiscribe RT enzyme 1 Water 7.32

The RT cycling reaction is composed of 10 uL test sample, 10 uL of RTprimer mix and 10 uL of RT Mix components for a total volume of 30 uL.The final concentration of the RT-primer in the total 30 uL total RT mixis 200 nM. The plate is then sealed with the same plate mat, brieflycentrifuged at 800 rpm for 1 minute, then run on the thermocycler usingthe following cycling parameters:

Cycling: 16° C. 30 minutes 42° C. 30 minutes 85° C.  5 minutes  4° C.hold

Next, 15 uL of Fast Enzyme/primer-probe mix is added to each well of anew Fast 96-well plate (Applied Biosystem's TaqMan Fast Universal PCRMaster Mix, Cat. No. 4352042)

ApoB PCR Master Mix Components uL/rxn Final Conc.Fast Enyzme Mix (2x stock) 10 forward primer (100 uM) 0.18 900 nMreverse primer (100 uM) 0.18 900 nM probe (10 uM) 0.05 250 nM Water 4.59ApoB primers and probe sequence: 17063DC F3 GGCGCGAAATTTCAGGAATTGT (SEQ.ID. NO.: 12) 17063DC Pr2 CACTGGATACGACCTTTAACA (SEQ. ID. NO.: 13)Universal R2 AGTGCAGGGTCCGAG (SEQ. ID. NO.: 14)

5 uL of each RT reaction is added to the Fast Enzyme Mix plate. Theplate is centrifuged for 1 minute at 1000 rpm and the QPCR analysis isperformed on an ABI7900 with Fast Block. Cycling parameters are: 1cycle—95° C. for 20 seconds, followed by 40 Cycles—95° C. for 1 seconds,60° C. for 20 seconds.

The QPCR result is utilized to calculate the siRNA concentration in thePEG antibody capture plate Triton lysates. Based on an estimate of 500siRNA per LNP particle, the number of LNPs retained in each well of theanti-PEG antibody plate can be calculated. Using the ApoE concentrationper well, as determined by the ApoE ELISA and the number of LNPparticles per well, an approximate ApoE molecules bound per LNP particlecan be calculated.

#ApoE Molecules Bound Per LNP

Compound ApoE Molecules/LNP 8 4.9 16 3.3 24 1.2 25 13.7 28 4.7 29 38 3212.8 33 18.1 34 2.3 45 (KC2)  32.5 46 (MC3) 14.5

Example 3 Heparin Sepharose HI-TRAP™ Binding Assay

Lipid nanoparticles (LNP) with neutral surface charge are not retainedafter injection onto heparin sepharose with 1× Dulbecco's phosphatebuffered saline (DPBS) as the running buffer but elute in the columnvoid volume. Serum apolipoprotein E (ApoE) exhibits high affinitybinding with heparin sulfate and it was shown that LNPs bind to heparinsepharose to an extent dependent on their intrinsic ability to bind ApoE(depending on both lipid nanoparticle composition and ApoEconcentration) after incubation with purified and/or recombinant humanApoE or serum samples. Lipid nanoparticles with surface bound ApoE bindto heparin sepharose with high affinity and are eluted only at high salt(1M NaCl).

A heparin sepharose binding assay was developed to assess serum ApoEbinding to lipid nanoparticles based on the high affinity interactionthat ApoE-LNP complexes exhibit toward heparin sepharose.

Incubations

Lipid nanoparticles were incubated at 37° C. for 20 min at a final siRNAconcentration of 50 μg/mL with various concentrations of either purifiedor recombinant human apolipoprotein E or 0.1-50% rat/mouse/rhesusmonkey/human serum in 1× Dulbecco's phosphate buffered saline (DPBS).After incubation with ApoE or serum LNP samples were diluted 10-foldusing 1×DPBS and analyzed by heparin sepharose chromatography. Peak areaof retained LNP (after subtraction of appropriate blank signals) iscompared to total peak area of LNP control without ApoE and/or serumincubation to determine the percentage of the LNP which undergoes shiftto high affinity heparin interaction after incubation with ApoE/serum.

Heparin Sepharose HI-TRAP™ Chromatographic Conditions

A heparin sepharose HI-TRAP™ chromatography column (GE Healthcare; 1 mLbed volume) is equilibrated with either 1× or 2× Dulbecco's PBS; thehigher 2× salt concentration is used for LNPs with higher intrinsicretention on heparin sepharose (presumably due to higher positivesurface charge).

Mobile Phase A: 1× or 2×DPBS

Mobile Phase B: 1M NaCl in 10 mM sodium phosphate buffer, pH 7.0

100% A delivered isocratically for 10 min followed by step gradient to100% B; hold for additional 10 min; step gradient back to 100% A andreequilibrate for additional 10 min prior to injection of next sample

Flow rate: 1 mL/minSample injection volume: 50 μL.

Detection: UV 260 nm HI-TRAP™ Binding Results Upon Rhesus SerumIncubation (2×DPBS Conditions)

Compound % Bound 32 100 33 <5 45 (KC2)  58 46 (MC3) 7

Example 4 Rat in Vivo Evaluation of Efficacy and Toxicity

LNPs utilizing compounds in the nominal compositions described above,were evaluated for in vivo efficacy and increases in alanine aminotransferase and aspartate amino transferase in Sprague-Dawley(Crl:CD(SD) female rats (Charles River Labs). The siRNA targets the mRNAtranscript for the ApoB gene (Accession # NM 019287). The primarysequence and chemical modification pattern of the ApoB siRNA isdisplayed above. The RDVs (containing siRNA) in PBS vehicle were tailvein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately3 ml/min. Five rats were used in each dosing group. After LNPadministration, rats are placed in cages with normal diet and waterpresent. Six hours post dose, food is removed from the cages. Animalnecropsy is performed 24 hours after LNP dosing. Rats are anesthetizedunder isoflurane for 5 minutes, then maintained under anesthesia byplacing them in nose cones continuing the delivery of isoflurane untilex-sanguination is completed. Blood is collected from the vena cavausing a 23 gauge butterfly venipuncture set and aliquoted to serumseparator vacutainers for serum chemistry analysis. Punches of theexcised caudate liver lobe are taken and placed in RNALater (Ambion) formRNA analysis. Preserved liver tissue was homogenized and total RNAisolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNAisolation kit following the manufacturer's instructions. Liver ApoB mRNAlevels were determined by quantitative RT-PCR. Message was amplifiedfrom purified RNA utilizing a rat ApoB commercial probe set (AppliedBiosystems Cat # RN01499054_ml). The PCR reaction was performed on anABI 7500 instrument with a 96-well Fast Block. The ApoB mRNA level isnormalized to the housekeeping PPIB (NM 011149) mRNA. PPIB mRNA levelswere determined by RT-PCR using a commercial probe set (AppliedBiosytems Cat. No. Mm00478295_ml). Results are expressed as a ratio ofApoB mRNA/PPIB mRNA. All mRNA data is expressed relative to the PBScontrol dose. Serum ALT and AST analysis were performed on the SiemensAdvia 1800 Clinical Chemistry Analyzer utilizing the Siemens alanineaminotransferase (Cat#03039631) and aspartate aminotransferase(Cat#03039631) reagents. Similar efficacy and improved tolerability wereobserved in rats dosed with Compound 32 or 33 containing RDV than withthe RDV containing the cationic lipid DLinKC2DMA (Compound 45) or MC3(Compound 46, FIG. 2).

Example 5 Determination of Cationic Lipid Levels in Rat/Monkey Liver

Liver tissue was weighed into 20-ml vials and homogenized in 9 v/w ofwater using a GenoGrinder 2000 (OPS Diagnostics, 1600 strokes/min, 5min). A 50 μL aliquot of each tissue homogenate was mixed with 300 μL ofextraction/protein precipitating solvent (50/50 acetonitrile/methanolcontaining 500 nM internal standard) and the plate was centrifuged tosediment precipitated protein. A volume of 200 μL of each supernatantwas then transferred to separate wells of a 96-well plate and 10 μlsamples were directly analyzed by LC/MS-MS.

Standards were prepared by spiking known amounts of a methanol stocksolution of compound into untreated rat liver homogenate (9 volwater/weight liver). Aliquots (50 μL) each standard/liver homogenate wasmixed with 300 μL of extraction/protein precipitating solvent (50/50acetonitrile/methanol containing 500 nM internal standard) and the platewas centrifuged to sediment precipitated protein. A volume of 200 μL ofeach supernatant was transferred to separate wells of a 96-well plateand 10 μl of each standard was directly analyzed by LC/MS-MS.

Absolute quantification versus standards prepared and extracted fromliver homogenate was performed using an Aria LX-2 HPLC system (ThermoScientific) coupled to an API 4000 triple quadrupole mass spectrometer(Applied Biosystems). For each run, a total of 10 μL sample was injectedonto a BDS Hypersil C8 HPLC column (Thermo, 50×2 mm, 3 μm) at ambienttemperature.

Mobile Phase A:

95% H2O/5% methanol/10 mM ammonium formate/0.1% formic acid Mobile PhaseB: 40% methanol/60% n-propanol/10 mM ammonium formate/0.1% formic acidThe flow rate was 0.5 mL/min and gradient elution profile was asfollows: hold at 80% A for 0.25 min, linear ramp to 100% B over 1.6 min,hold at 100% B for 2.5 min, then return and hold at 80% A for 1.75 min.Total run time was 5.8 min. API 4000 source parameters were CAD: 4, CUR:15, GS1: 65, GS2: 35, IS: 4000, TEM: 550, CXP: 15, DP: 60, EP: 10.

In rats dosed with Compound 32 or 33 containing RDV, liver levels wereeither similar to or lower than the RDV containing the cationic lipidDLinKC2DMA (Compound 45) or MC3 (Compound 46, FIG. 3). In monkeys dosedwith Compound 32 or 33 containing RDV, liver levels were lower than theRDV containing the cationic lipid DLinKC2DMA (Compound 45) or MC3(Compound 46, FIG. 7).

Example 6 Rhesus Monkey in Vivo Evaluation of ApoB Efficacy

LNPs utilizing compounds in the nominal compositions described above,were evaluated for in vivo efficacy in male or female Macaca mulatta(rhesus) monkeys. The siRNA targets the mRNA transcript for the ApoBgene (Accession # XM 001097404). The primary sequence and chemicalmodification pattern of the ApoB siRNA is displayed above. The RDVs(containing siRNA) in PBS vehicle were administered by intravenousinjection in the saphenous vein at an injection rate of 20 mL/minute toa dose level of 0.25 mg/kilogram siRNA. The injection volumes were from1.9 to 2.1 mL/kilogram and monkeys ranged in weight from 2.5 to 4.5kilograms. The RDV or PBS control were administered to three monkeys. Atmultiple days post dose, 1 mL blood samples were drawn from the femoralartery for serum chemistry analysis. Monkeys were fasted overnight priorto blood draws. As a measure of efficacy, LDL-C was monitored as adownstream surrogate marker of ApoB mRNA reduction. At 4 days postsystemic administration of RDVs containing compounds 32 and 33 (0.25mg/kg), serum levels of LDL-C were reduced to less than 30% of pre-doselevels (FIG. 4).

Example 7 Rhesus Monkey in Vivo Evaluation of β-Catenin Efficacy

On study day −7 predose liver biopsy samples (˜0.5-1 gram/sample) werecollected from male rhesus monkeys by laparoscopic surgical resection(resection of one biopsy sample from outer edge of one randomly selectedliver lobe per monkey). A 5 mm tissue punch was used to sample threenon-adjacent ˜50 mg samples from each predose biopsy. Samples werepreserved in RNAlater™ (Ambion) for later CTNNB1 mRNA analysis.

On study day 0 monkeys were administered suspensions of the lipidnanoparticle (LNP) test articles in phosphate buffered saline (0.05-0.1mg siRNA/mL) via single-dose intravenous bolus injection at target dosesof 0.67, 1.34 or 3.34 mg siRNA/m². For dosing purposes, body surfacearea (m²) was estimated from body weight according to the establishedallometric scaling relationship given below (1):

BSA(m²)=0.11*BW(in kg)^(0.65)

On study days 2 and 7, at 48 hours and 168 hrs post LNP administration,liver biopsy samples (˜0.5-1 gram/sample) were collected from monkeys bylaparoscopic surgical resection (2 separate randomly selected liverlobes were resected per monkey). A 5 mm tissue punch was used to samplethree non-adjacent ˜50 mg samples per each 48 hr and 168 hr surgicalbiopsy sample. Samples were preserved in RNAlater™ (Ambion) for laterCTNNB1 mRNA analysis.

CTNNB1 mRNA levels were measured by relative quantitative RT-PCR using aprimer/probe set validated for CTNNB1 and normalized against mRNA levelsof peptidylprolyl isomerase B (also known as PPIB or cyclophilin B) andRNA levels of 18S ribosomal RNA (18S rRNA). Change in CTNNB1 mRNA liverexpression was measured as the difference in PCR threshold cycle number(ΔΔCt) between post-dose samples and each corresponding monkey's predoseliver samples.

Calculation of CTNNB1 mRNA knockdown (with respect to pretreatmentlevels) was calculated from ΔΔCt using the following relationship:

mRNA(% knockdown)=100−(100/2^(−ΔΔCt))

Monkeys dosed with RDVs containing compounds 32 and 33 and beta-cateninsiRNA demonstrated robust KD at doses ranging from 0.67-3.34 mg/m² (FIG.5).

(1) FDA Guidance Document: “Guidance for Industry: Estimating theMaximum Safe Starting Dose in Initial Clinical Trials for Therapeuticsin Adult Healthy Volunteers” July 2005, US Department of Health andHuman Services, Food and Drug Administration—Center for Drug Evaluationand Research (CDER)

Example 8 Rhesus Monkey in Vivo Evaluation of ALT Increases

Alanine aminotransferase (ALT) is measured in serum that is harvestedfrom clotted monkey whole blood after centrifugation. A Roche ModularSystem automated chemistry analyzer measures the enzymatic activity ofALT in the serum by using International Federation of Clinical Chemistrystandardized procedures and reagents. The analyzer's computer usesabsorbance measurements to calculated ALT activity in the sample ascompared to a standard curve. The ALT activity is reported inInternational Units per Liter (IU/L).

Monkeys dosed with RDVs containing compounds 32 and 33 had lower peakALT elevations than those dosed with the RDV containing the cationiclipid DLinKC2DMA (Compound 45) or MC3 (Compound 46, FIG. 6).

Example 9 Evaluation in Hepatocellular Carcinoma Mouse Model

The activity LNPs in delivering a β-catenin siRNA (siRNAβ-cat) tohepatocellular carcinoma was evaluated in a mouse hepatocellularcarcinoma (HCC) model, named TRE-MET. TRE-MET mice are transgenic micein an FVB/N genetic background where the human MET transgene isexpressed under an hCMV promoter with heptamerized upstreamtet-operators. When TRE-MET mice are crossed with the LAP-tTA line, thedouble transgenic (TRE-MET/LAP-tTA) mice express MET in a liver-specificmanner which can be suppressed by administration of doxycycline. Thesemice develop HCC at ˜3 months of age with visually identifiable tumornodules on the liver surface and the tumors display a diffuse trabeculargrowth pattern typical for HCC and express the HCC tumor markeralpha-fetoprotein (AFP). In addition, the mutation analysis in the tumorof TRE-MET mice has also identified activating mutations in thebeta-catenin gene in approximately 95% of tumors. These features makethe TRE-MET HCC mouse model suitable for evaluating LNP-mediateddelivery of β-catenin siRNA and the resultant efficacy on tumor growth.

The effect of β-catenin containing LNPs in silencing β-catenin mRNA inboth liver and tumor tissues was first evaluated in a pharmacodynamic(PD) study in TRE-MET mice bearing tumors. Different doses of LNPs or ahigh dose of LNP control siRNA were intravenously administered and 72hours later, necropsy was performed to collect liver and tumor tissuesfor the determination of β-catenin mRNA levels by Taqman. As shown inFIGS. 8 and 9, Compound 33 induced robust and dose-dependent knockdownof β-catenin mRNA in both liver and tumor tissues, whereas no β-cateninknockdown was observed in animals receiving control siRNA or PBS. 0.1mpk and 0.05 mpk of LNP induced 88% and 69% KD in normal liverrespectively. The KD in tumors ranges from 70% (2 mpk) to about 40% (0.1or 0.05 mpk). As shown in FIGS. 12 and 13, Compound 32 induced robustand dose-dependent knockdown of β-catenin mRNA in both liver and tumortissues, whereas no β-catenin knockdown was observed in animalsreceiving control siRNA or PBS. 0.1 mpk and 0.05 mpk of LNP induced 76%and 78% KD in normal liver respectively. The KD in tumors ranges from47% (0.25 mpk) to about 20-30% (0.1 or 0.05 mpk).

The effect of LNP on tumor growth was evaluated in a multiple-doseefficacy study. TRE-MET HCC mice were dosed with Compound 33/siRNAβ-cat,Compound 32/siRNAb-cat, control siRNA or PBS weekly for 3 weeks (3doses) and the tumor volume in each animal was determined 7 days priorto the 1^(st) dose and 3 days post the final dose by microCT scan (FIGS.10 and 14). In addition, 7 days after the final dose, liver and tumortissues were collected for the assessment of β-catenin mRNA levels.While mice receiving PBS or control siRNA showed 360-470% growth intumor burden, mice treated with Compound 33/siRNAβ-cat exhibitedprofound tumor growth inhibition or regression in a dose-dependentmanner (FIG. 10). 2 mpk and 0.5 mpk of Compound 33/siRNAβ-cat induced60% and 40% tumor regression respectively and 0.05 mpk caused tumorstasis. While mice receiving PBS or control siRNA showed ˜350% growth intumor burden, mice treated with Compound 32/siRNAβ-cat exhibitedprofound tumor growth inhibition or regression in a dose-dependentmanner (FIG. 14). 0.5, 0.25 and 0.1 mg/kg of Compound 32/siRNAβ-catinduced 37, 58, and 37% tumor regression respectively and 0.05 mpkcaused tumor stasis.

1. A cationic lipid of Formula A:

wherein: R¹ and R² are independently selected from H, (C₁-C₆)alkyl,heterocyclyl, and polyamine, wherein said alkyl, heterocyclyl andpolyamine are optionally substituted with one to three substituentsselected from R′, or R¹ and R² can be taken together with the nitrogento which they are attached to form a monocyclic heterocycle with 4-7members optionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclicheterocycle is optionally substituted with one to three substituentsselected from R′; R³ is independently selected from H and (C₁-C₆)alkyl,said alkyl optionally substituted with one to three substituentsselected from R′; R′ is independently selected from halogen, R″, OR″,SR″, CN, CO₂R″ or CON(R″)₂; R″ is independently selected from H and(C₁-C₆)alkyl, wherein said alkyl is optionally substituted with halogenand OH; n is 0, 1, 2, 3, 4 or 5; L₁ is selected from C₄-C₂₄ alkyl andC₄-C₂₄ alkenyl, said alkyl and alkenyl are optionally substituted withone or more substituents selected from R′; and L₂ is selected from C₃-C₉alkyl and C₃-C₉ alkenyl, said alkyl and alkenyl are optionallysubstituted with one or more substituents selected from R′; or anypharmaceutically acceptable salt or stereoisomer thereof.
 2. A cationiclipid of Formula A according to claim 1, wherein: R¹ and R² are eachmethyl; R³ is H; n is 0; L₁ is selected from C₄-C₂₄ alkyl and C₄-C₂₄alkenyl; and L₂ is selected from C₃-C₉ alkyl and C₃-C₉ alkenyl; or anypharmaceutically acceptable salt or stereoisomer thereof.
 3. A cationiclipid of Formula A according to claim 1, wherein: R¹ and R² are eachmethyl; R³ is H; n is 2; L₁ is selected from C₄-C₂₄ alkyl and C₄-C₂₄alkenyl; and L₂ is selected from C₃-C₉ alkyl and C₃-C₉ alkenyl; or anypharmaceutically acceptable salt or stereoisomer thereof.
 4. Thecationic lipid according to claim 1 which is selected from:(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine (Compound 1);(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-9-amine (Compound 2);(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-8-amine (Compound 3);(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine (Compound 4);(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine (Compound 5);(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine (Compound 6);(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine (Compound 7);(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine (Compound 8);(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine (Compound 9);(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine (Compound 10);(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-9-amine (Compound 11);(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine (Compound 12);(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine (Compound 13);(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine (Compound 14);(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine (Compound 15);(21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine (Compound 16);(18Z)—N,N-dimethylheptacos-18-en-10-amine (Compound 17);(17Z)—N,N-dimethylhexacos-17-en-9-amine (Compound 18);(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine (Compound 19); andN,N-dimethylheptacosan-10-amine (Compound 20);(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine (Compound 21);1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine (Compound 22);(20Z)—N,N-dimethylheptacos-20-en-10-amine (Compound 23);(15Z)—N,N-dimethylheptacos-15-en-10-amine (Compound 24);(14Z)—N,N-dimethylnonacos-14-en-10-amine (Compound 25);(17Z)—N,N-dimethylnonacos-17-en-10-amine (Compound 26);(24Z)—N,N-dimethyltritriacont-24-en-10-amine (Compound 27);(20Z)—N,N-dimethylnonacos-20-en-10-amine (Compound 28);(22Z)—N,N-dimethylhentriacont-22-en-10-amine (Compound 29);(16Z)—N,N-dimethylpentacos-16-en-8-amine (Compound 30);(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (Compound 31);(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32);N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (Compound33); 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine(Compound 34);N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine (Compound35); N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine(Compound 36);N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine(Compound 37);N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine (Compound38); N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine(Compound 39);N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine(Compound 40)1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine (Compound41); 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine(Compound 42);N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine (Compound43); and (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,23-trien-10-amine(Compound 44); or any pharmaceutically acceptable salt or stereoisomerthereof.
 5. The cationic lipid according to claim 1 which is:(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32);or any pharmaceutically acceptable salt or stereoisomer thereof.
 6. AnLNP composition which comprises, a cationic lipid of Formula A accordingto claim 1, cholesterol, DSPC and PEG-DMG.
 7. An LNP composition whichcomprises, the cationic lipid(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32),cholesterol, DSPC and PEG-DMG.
 8. The use of a cationic lipid accordingto claim 1 for the preparation of lipid nanoparticles.
 9. The use of acationic lipid according to claim 1 as a component in a lipidnanoparticle for the delivery of oligonucleotides.
 10. The use accordingto claim 9 wherein the oligonucleotides are siRNA.